A skin wound is defined as a breach in the continuity of any body tissue caused by a minimal direct injury to the skin. There are many instances where a quick closure of the wounded skin will promote a beneficial response. Generally, quick closure of wounded skin can be achieved either by conservative methods such as the application of medicaments, or alternatively, by using various surgical procedures including suturing, split skin grafting or grafting of new skin grown in culture.
The closure of a wound with skin cells is performed using two methods: either by grafting skin grown in culture or alternatively, by split skin grafting. These two methods are applicable, however, only after a suitable base of granulation tissue has first developed in the wound, the development of which may be quite prolonged or complicated. Split skin grafting, although more common, requires compositions which contain materials for maintaining organ viability and treatment of the wounds for the repair of injury to the skin.
Among the most common injuries to skin are burns. Burn causes destruction of the epidermis and deeper cutaneous and subcutaneous tissues, most of which can be regenerated by the normal healing response if the area burned is not extensive or contaminated. Burns cause more than 2,000,000 injuries annually in the U.S.A., and more than 10,000 deaths each year result from serious burn injuries.
S. T. Boyce et al., in The Journal of Investigative Dermatology, 81: 33S-40S, 1983) describes compositions based upon a serum-free culture system to culture normal human epidermal keratinocytes. These compositions comprise optimized nutrient medium MCDB 153 supplemented with epidermal growth factor, insulin, hydrocortisone, ethanolamine, phospho-ethanolamine and whole Bovine Pituitary Extract (wBPE). It is mentioned that the wBPE initiates the primary culture and that cellular senescence occurs after about forty population doublings. It has also been reported in the Journal of Cellular Physiology, 110, 219, (1982), that the incorporation of Fetal Bovine Serum Protein (FBSP) may replace whole serum for culturing human epidermal keratinocytes and that the presence of F12 would eliminate the need for wBPE. As presently known, wBPE is not a common reagent which can be easily reproducibly prepared, its constitution not being constant.
J. J. Wille, Jr. et al., in the Journal of Cellular Physiology, 121, 31, (1984) describes the effects of growth factors, hormones and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes. Clonal growth was achieved when MCDB 153 was supplemented with epidermal growth factor or wBPE, provided that insulin was present. In the absence of insulin both EGF and wBPE are required. It is mentioned that optimal clonal growth occurred in medium containing 10 ng/ml of epidermal growth factor and 0.3 mM calcium.
According to U.S. Pat. No. 4,673,649, compositions are suggested for clonal growth of a population of human keratinocyte cells in a primary culture for the repair of injury to skin, having a characteristic colony-forming efficiency of about 20%. The composition comprises: MCDB 153, epidermal growth factor a concentration range of 1.0 ng/ml to 25 ng/ml and insulin at a concentration range of 0.5 ug/ml to 50 ug/ml. Optionally, the compositions may contain wBPE (whole bovine pituitary extract) at a concentration range of 7 ug/ml to 700 ug/ml, ethanolamine, hydrocortisone, phosphoethanolamine and calcium chloride. In particular, the compositions are useful for growing skin cells for grafting. No mention is made of the possible use of the disclosed compositions to treat wound conditions in vivo, nor to prolong and preserve the viability of stored split skin grafts. In a very recent U.S. Pat. No. 4,940,666 (by the same inventors and as a c.i.p. of the previous U.S. Patent), the same compositions are claimed to be useful for growing a population of human epidermal cells. The purpose of the compositions suggested is for the propagation of skin cells and achieving monolayers, or stratified layers, of keratinocytes to be used for areas on the body without skin. In other words, these compositions are used for the development of cultured skin cells which may be used for grafting. In addition to the above references, other prior art references suggest that epidermal growth factor may enhance wound healing by increasing fibroblast proliferation.